Some laboratory experience required: knowledge of sterile technique, working with bacterial cultures, and using automatic pipets all helpful.

Material Availability

Specialty items

Cost

Average ($50 - $100)

Safety

Requires adult supervision in a laboratory facility. For ISEF-affiliated fairs, this project will require SRC approval.

Abstract

Is it possible to manipulate bacteria to become protein production factories? Can diabetics control blood glucose with insulin produced by bacteria? How cool would it be to take advantage of these microorganism's sophisticated makeup, short doubling times and cheap growth media to mass produce medically and commercially useful proteins? All of these are possible with a few simple genetic manipulations. By the end of this project you would know the basic foundation on which many biotechnology and pharmaceutical companies operate.

Objective

The goal of this project is to measure bacterial transformation efficiency as a function of plasmid DNA concentration.

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Introduction

Bacteria are biochemical powerhouses, completely self-contained with all that is needed to produce complex proteins. Without microscopes, the human eye only begins to see bacteria when they have multiplied to literally millions of identical copies all in one spot, called a colony. The DNA molecule is the blueprint for every component of the bacteria. From information in the DNA, RNA molecules are transcribed and then translated into proteins. The proteins are moved to different parts of the bacteria—cytoplasm, periplasm, or cell wall—depending on function. Bacteria have transport systems that shuttle these proteins around, and sometimes there may be more than one type of transport system.

There are several ways in which bacteria acquire foreign DNA, including the processes of conjugation, transfection, and transformation. Conjugation involves mating between two different bacterial cells. In transfection, viruses called bacteriophages inject the foreign DNA into their host. In transformation, bacteria take up DNA from the environment through their cell wall.

Natural transformation was discovered in 1928 by Frederick Griffith while studying infectious bacteria that cause pneumonia in mice. Griffith was using two strains of pneumococcus bacteria: a virulent, smooth strain and a non-virulent, rough strain. On injecting mice with a mixture of killed smooth strains (which were incapable of causing infection) and the non-virulent rough strain, miraculously (the miracle being what is now known as transformation), the mice died and Frederick recovered live smooth pneumococcus bacteria! Inside the mice, the piece of DNA from the killed smooth strain containing the information required to cause infection had been taken up by the live, non-virulent, rough pneumococcus bacteria. In this way, the formerly non-virulent bacteria acquired the virulence traits and became deadly to mice. In Frederick's control experiments, mice exposed to only rough cells stayed nice and healthy and those exposed to the dead smooth cells also stayed nice and healthy. Only when the live rough bacteria and killed smooth bacteria were administered together did the mice become sick.

Why Is Bacterial Transformation So Important and Why Does It Hold Such Attraction to Science?

Transformation in and of itself is a very important basic tool in molecular biology. Transformation is used for cloning or to move DNA molecules around between strains. Bacteria are transformed for numerous different reasons. Some of these reasons may include expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease. Other reasons could be expression of proteins that confer on bacteria the ability to survive in particular environments such as to "clean up" contaminated environments in bioremediation.

It can be very expensive to chemically synthesize very short peptides, never mind complex polypeptides and whole proteins which may have post-translational modifications. As the biology of bacteria becomes clearer, coupled with the abundance of bacterial species and strains available and the exciting advances made in molecular biology research and biotechnology, the possibilities and applicability of transformation becomes phenomenal.

How Does It Work?

Not all bacteria undergo natural transformation. However, a large number of strains and species can be artificially transformed. In the laboratory when transformation occurs, the bacteria acquire new genetic traits (for example, resistance to a specific antibiotic) which are easily identifiable and allow for selection of transformed cells.

Before bacteria can be artificially transformed, they have to be made competent—able to take up DNA. The DNA molecule is hydrophilic (water-soluble) but cell membranes are made of a very hydrophobic lipid bilayer, and therefore artificial transformation is not a process that occurs spontaneously. There are two means of artificial transformation commonly used in labs: electroporation and chemical transformation.

During electroporation, short bursts of current are passed through a solution containing bacteria at high voltage. The current makes the cell membrane leaky (porous) for a short time, allowing the cells to take up DNA molecules from the solution.

In chemical transformation, bacteria are exposed to solutions which alter their cell membranes enough to make the DNA molecules pass through and into the cell. Chemical transformation procedures sometimes also use a heat shock treatment. The actual mechanisms by which these two processes work are not fully understood.

Transformation efficiency is a measure of the amount of cells within the bacterial culture that are able to take up DNA molecules. Transformation efficiency can be determined experimentally. For some molecular biology projects, such as cloning and subcloning, high transformation efficiency is not critical. However applications such as construction of genomic libraries require that the bacteria have very high transformation efficiency.

When bacteria are transformed in the laboratory, the bacteria acquire new traits from the transformation plasmid. These traits are easily identifiable and allow for selection of transformed cells. For example, the bacteria transformed in this project acquire resistance to the antibiotic ampicillin. You prove this by growing them up on LB:AMP plates. Untransformed bacteria will not grow on the LB:AMP plates.

Sometimes, scientists get really creative and add genes for other traits to these plasmids. Take the pGLO plasmid from the Bio-Rad pGLO transformation kit as an example. In addition to containing the ori gene which is essential for the plasmid to replicate inside the bacteria, and the Ampicillin resistance gene (bla gene) used for selection of transformed cells, it also contains the gene that encodes the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. The GFP gene is placed under the regulation of the arabinose promoter. Because bacteria are highly economical and tend not to waste energy, they have different types of promoters which regulate gene expression. For those proteins which are required only under specific conditions, the genes that encode them are turned off when not needed to conserve energy and turned on only when the bacteria encounters the right conditions. For example, the genes which encode the proteins required to break down and use the sugar arabinose are turned on only in the presence of arabinose. Therefore by placing the GFP protein gene under the control of the arabinose promoter, scientists can selectively express the GFP protein by including or excluding arabinose from the growth media. This allows for increased utility of the pGLO plasmid not just simply for cloning, but also for in situ visualization of protein expression because those bacteria that have taken up the pGLO plasmid will turn a brilliant green color (as shown in Figure 1, below) when they are grown in a media containing arabinose.

This is a simplified version of the classical method used in this transformation exercise (see the Variations section):
Chung, C.T., S.L. Niemala, and R.H. Miller, 1989. "One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution," PNAS 86 (7): 2172–2175, available online [accessed October 20, 2006]: http://www.pnas.org/cgi/reprint/86/7/2172 (requires Adobe Acrobat).

At the Bio-Rad website, you can download the complete instruction manual for the pGLO transformation Kit. The manual contains lots of useful information including diagrams and protocols (requires Adobe Acrobat). In order to download literature, you must register at the Bio-Rad site,
and then search for "pGLO" in the "Literature" section of the search box.:
Bio-Rad, 2006. "pGLO Bacterial Transformation Kit Instruction Manual," Bio-Rad, Inc. [accessed October 25, 2006] http://explorer.bio-rad.com.

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Materials and Equipment

Note: If you are carrying out this experiment in a school laboratory, which is recommended, some of the materials and equipment listed may be more readily accessible.

The pGLO Bacterial Transformation Kit from Bio-Rad is recommended however, any
equivalent transformation kit can be used. The pGLO transformation kit contains
the bacteria, plasmid DNA, reagents, etc. A teacher's help will be needed when
ordering the pGLO kit as Bio-Rad only sells directly to schools.

Permanent marker

Ice bucket with ice. You can substitute styrofoam cups with ice.

37°C incubator. Alternatively, bacteria will grow at room temperature but it takes days longer.

42°C water bath. Alternatively, hot water from the tap is sufficient; use a thermometer to ensure the temperature is exactly 42°C for the 50 second duration of the heat shock.

Automatic pipettors in 1–10 μL and 10–200 μL ranges. Graduated pipets supplied with the Bio-Rad kit can be used instead if unavailable.

UV-protective face shield or goggles for visualizing pGLO. UV-protective goggles are available through Carolina Biological.

Note: Bio-Rad Kits are sold directly to schools. To purchase, please have your school contact Bio-Rad at 800-424-6723 to verify account information and to place the order for you. Existing accounts will have orders processed within a day, and establishing an account will take approximately 48 hours.

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Experimental Procedure

Working with Biological Agents

For health and safety reasons, science fairs regulate what kinds of biological materials
can be used in science fair projects. You should check with your science fair's
Scientific Review Committee before starting this experiment to make sure your science
fair project complies with all local rules. Many science fairs follow Intel®
International Science and Engineering Fair (ISEF) regulations. For more information,
visit these Science Buddies pages:
Projects Involving Potentially Hazardous Biological Agents and
Scientific Review Committee. You can also visit the webpage
ISEF Rules & Guidelines directly.

Do your background research so that you are knowledgeable about the terms, concepts, and questions above.

Read the product insert from the transformation kit prior to starting your experiment.

You need to understand the sequence of the experimental protocol and prepare materials accordingly.

Follow the directions provided in the kit. Note that you will include an additional step to determine how varying the concentration of DNA affects transformation efficiency.

The following procedure is based on the assumption that the Bio-Rad pGLO transformation kit is used.

Day 1: Preparing Plates, Solutions, and Bacterial Starter Plate

Follow the kit directions to prepare and pour the agar plates, and to rehydrate the provided lyophilized materials such as E. coli bacteria, antibiotics, DNA, etc.

Prepare and pour the agar plates—LB only (LB) and LB plus ampicillin (LB:AMP).

Label the plates with permanent marker: LB and LB:AMP.

After the agar solidifies, cover the plates, put them in their original plastic bags, and store them in a lab refrigerator stacked upside down. Store plates wrapped up in their original plastic wrappings. Storing upside down will ensure condensation does not wet the surface of the agar.

Note that the pGlo Transformation kit also allows for visualization of the transformation. In addition to the acquisition of Ampicillin resistance, the transformed bacteria can also express another gene on the pGLO plasmid which causes the bacteria to glow a brilliant green color. In order to see this, prepare the LB:AMP:ARA agar plates as specified in the pGLO transformation kit product insert. After transformation on day 2, plate the transformed cells on the LB:AMP:ARA plates as well. The arabinose in the agar will induce expression of the green fluorescent protein and the bacteria will glow green. While this step is cool to see it is not required for you to determine transformation efficiency. The Bio-Rad pGLO transformation kit comes with one UV penlight. This should be sufficient to visualize the glowing bacteria. However if you have access to a laboratory with a long wave UV lamp, that will be great. Caution - Do not shine UV light directly into the eyes, use a UV-protective face shield or goggles, and limit exposure to UV light.

Rehydrate bacteria and streak LB starter plates.

Incubate starter plates overnight at 37°C (or 2 to 3 days at room temperature until colonies are clearly visible).

Day 2: Transforming the Bacteria

Important: do not refrigerate the starter plate prior to transformation.

Label 3 tubes with the following:

−pGLO plasmid (negative control),

+1× pGLO plasmid,

+10× pGLO plasmid.

Using a graduated pipette add 1 mL of the transformation solution to a clean tube.

With a sterile loop, choose 4 well-separated colonies from the starter plate and resuspend in the tube containing 1 mL of transformation solution by flicking the tube or by twirling the loop around in the solution. (Note that each colony contains millions of bacterial cells—do not use too many colonies).

When the colonies are completely resuspended, use a clean graduated pipette to transfer 250 μL of the bacterial solution to each of the 3 tubes labeled −pGLO plasmid, +1× pGLO plasmid, and +10× pGLO plasmid.

Add 10 μL of the pGLO plasmid solution to the tube labeled +1× pGLO plasmid. (If you don't have access to automatic pipettors, 10 μL is one sterile loop full.)

Calculate transformation efficiency for the 1X and 10X DNA concentrations using the formula.

Which experiment had greater efficiency?

Bacterial Safety

Bacteria are all around us in our daily lives and the vast majority of them are
not harmful. However, for maximum safety, all bacterial cultures should always be
treated as potential hazards. This means that proper handling, cleanup, and disposal
are necessary. Below are a few important safety reminders. You can also see the
Microorganisms Safety
Guide for more details. Additionally, many science fairs follow
ISEF Rules & Guidelines, which have specific guidelines on how bacteria
and other microorganisms should be handled and disposed of.

Keep your nose and mouth away from tubes, pipettes, or other tools that come in
contact with bacterial cultures, in order to avoid ingesting or inhaling any bacteria.

Make sure to wash your hands thoroughly after handling bacteria.

Proper Disposal of Bacterial Cultures

Bacterial cultures, plates, and disposables that are used to manipulate the bacteria
should be soaked in a 10% bleach solution (1 part bleach to 9 parts water)
for 1–2 hours.

Use caution when handling the bleach, as it can ruin your clothes if spilled, and
any disinfectant can be harmful if splashed in your eyes.

After bleach treatment is completed, these items can be placed in your normal household
garbage.

Cleaning Your Work Area

At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach
solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly
clean any surfaces you have used.

Be aware of the possible hazards of disinfectants and use them carefully.

Recent Feedback Submissions

What was the most important thing you learned?
I learned that the more bacteria you insert into a cell, it could affect the growth of the bacteria or even overwhelm the cell.

What problems did you encounter?
The biggest problem in this experiment was finding a lab and a mentor. After over 50 emails, I got acceptance from a microbiologist.

Can you suggest any improvements or ideas?
If you do this experiment, make sure to do more than 1 trial. Also test if heat shock effects the experiment also. I only used 1 trial, so I don't know if my results were accurate or not. So in the future that's what I could look into. Also if you would like to do this experiment, use all the concentrations ranging from 1 to 10. In my experiment, I only used 1 and 10, which is a huge gap. So In the future, I can test the optimal range of concentration.

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